The serum-free culture of normal human epidermal keratinocytes without the use of companion-cells,, cell feeder layer or organotypic substrate, e.g. collagen or gelatin, is disclosed in this invention. Traditionally, tissue culture of normal epithelial cells has been attempted in a variety of commercially available media designed for the growth of less fastidious types of cells, i.e., malignant cells transformed in vitro from cell lines derived from human or nonhuman tissues, cell lines developed from human or nonhuman tumors, or cell lines developed for human or nonhuman embryonic mesenchymal cell types. In contrast, the culture of normal human epithelial stem cells has presented many difficulties not the least of which is the inexorable tendency for these cells to undergo uncontrolled, irreversible, terminal differentiation with the consequent loss of cell division capacity. A significant development which permitted the growth of human epidermal cells in culture was the formulation of a selective basal nutrient medium and its supplementation with specified growth factors and hormones [Tsao, M. C., et al., J. Cellular Physiol. 110:219-229 (1982)]. This selective medium was designated MCDB 152. Further refinements of this medium lead to the formulation of MCDB 153 [Boyce, S. T. and Ham, R. G., J. Invest. Dermatol. 81:33-40 (1983) ]. The use of these media permitted a more accurate characterization of the necessary growth factors, hormones and Ca.sup.2+ requirements for retention of high cloning efficiency necessary to maintain proper genetic programming for continued subculture of pluripotent basal epidermal stem cells [Wille, J. J., et al., J. Cellular Physiol. 121:31-44 (1984)].
The actual role of serum in cell culture medium as a complex mixture of both growth factors and differentiation-inducing factors was resolved by careful clonal growth, cell division kinetics, and flow cytofluorography [Pittelkow, M. R., et al., J. Invest. Dermatol. 86:410-417 (1986) ]. These findings indicated that serum, known to contain fibroblastic cell growth factors, e.g.,, 5 platelet-derived growth factor, was an inhibitor of basal epidermal cell growth. Further, the differentiation-inducing factors in serum could be equated with serum's content of 8-transforming growth factor, (.beta.-TGF) , [Shipley, S. D., et al., Cancer Res. 46:2068-2071 (1986)]. Recently, the inventor and colleagues reported that normal human keratinocytes actually produce their own growth factors. That is, proliferating basal cells are stimulated to secrete .alpha.-transforming growth factor (.alpha.-TGF) in response to the presence of added epidermal growth factor (EGF) and decrease production of .alpha.-TGF at high cell densities near confluence. Under the latter condition, the arrested cells secrete an inactive form of .beta.-TGF [Coffen, R. J., et al. , Nature 328:817-820 (1987)]. These considerations recently led the inventor to the idea that the natural mechanism of growth stimulation and its regulation in intact epidermis involved coordinated secretions of .alpha.- and .beta.-TGF's, and that the provision of such factors would eliminate the need for any organic substrate as well. Further experimentation to verify this surmise resulted in the findings in the present invention.
Previously, a patent [Green, H. and Kehinde, O., U.S. Pat. No. 4,304,866, 1981] was obtained for an in vitro method for the formation of epithelial sheets from cultured keratinocytes. This method uses a serum-containing medium and a feeder layer of murine (mouse) fibroblast cells to accomplish cell growth and differentiation. This procedure has serious limitations for large scale production of genetically-defined (autologous) human skin substitute. For example, the use of serum inextricably confounds the culture of purely basal cells with the dynamics of serum-induced differentiation. The net result is that subcultivation of such cultures yields low (&lt;5%) clonal efficiencies preventing step-wise large scale build up of uncommitted pluripotent basal cells as a prelude to their conversion into usable sheets of transplantable, histologically-complete, human epidermis. moreover, the process of Green, H. and Kehinde, O. [U.S. Pat. No. #4,304,866, 1981] does not describe a histologically-complete epidermis. i.e. an epidermis which is formed of all six major identifiable layers of a complete human epidermis. Rather, the procedures therein can only form an epidermis lacking a stratum corneum (SEE: FIG. 1E], this being necessary for maximizing the utility of the tissue, and, thus, this limits the product uses. In a more recent methodology, a complete epidermis has been achieved, but only in the presence of a complete skin starter sample and serum-containing media that are combined with an organotypic substratum containing growth factors produced by companion cells [E. Bell, U.S. Pat. No. 4,485,096, 1984; and E. Bell & L. Dubertret, U.S. Pat. No. 4,604,346, 1986]. Although it is conceivable that these latter processes may be used in the absence of serum, the continued use of any organotypic substrate as well as feeder or companion cell types, e.g. fibroblasts, seriously limits, in an immunologically safe manner as well as an economic manner, their large-scale use, e.g. burn patients [Nanchahal, J. , et al., Lancet II (8656) :191-193, (1989)]. In order to remedy these deficiencies the inventor has dispensed with serum-containing media, eliminated any substratum support, dispensed with the requirement for innumerable skin starter samples, and designed a new basal nutrient medium capable of supporting the growth and development of a complete epidermis. Moreover, the identification of essential process steps leading to a functional epidermis has been discovered and can be monitored with specific monoclonal antibodies. In retrospect, the culturing of epidermal keratinocytes in medium containing undefined serum and/or feeder cell factors and/or organotypic substrates, and millimolar concentrations of ca 21 were not designed for the unlimited proliferation of undifferentiated basal cells. Such cultures can spontaneously undergo maturation and uncontrolled differentiation. The result was that an incomplete epidermis was produced. By contrast, the design of serum-free culture process described in this invention produces a complete epidermis by an orderly sequence at will, from a defined starting point in the culture process.